Inactivated virus compositions and methods of preparing such compositions

ABSTRACT

The present invention is directed at a composition comprising a live swine flu virus having an infectious component and a plurality of surface antigens in contact with a formaldehyde donor agent having a molecular weight that is less than about 400 g/mol. Preferably, the formaldehyde donor agent is selected from a non-crosslinking chemical fixative that contains urea.

CLAIM OF PRIORITY

This application claims priority from provisional application Ser. No.61/482,367 filed May 4, 201 incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates generally to inactivated swine flu viruscompositions, methods of preparing such compositions, and to suchcompositions useful as or in vaccines. Further the present inventionrelates to methods of preparation, storage and transportation ofprecursors to and vaccine compositions wherein the precursors andvaccines can be stored and transported at ambient temperatures.

BACKGROUND OF THE INVENTION

It is known that the preparation of vaccines commonly involves the stepsof inactivating a live virus (whether it be attenuated, modified orkilled), so that the virus thereafter can be introduced into a livingbeing for inducing an active immune response (e.g., a protectiveresponse) in the being. The manufacture of such vaccines oftenencounters any of a number of practical constraints, which create astrain on health care systems. By way of example, in recent history,shortages of influenza vaccines have been experienced due tocontamination problems in the manufacturing process. In the context ofviral vaccines, one approach to the manufacture of such vaccines hasbeen to grow the specific virus in advance of preparing the vaccinecomposition, such as by growing the virus in chicken eggs. Themanufacture of viruses according to that approach can be expensive, andtime consuming, especially considering that many times, one or two eggsmay be necessary to yield each dose of vaccine. One classic approach tothe manufacture of vaccines has been to inactivate a virus withformalin, binary ethylenimine, formaldehyde or combinations thereof. Itis also typically necessary in the manufacture of vaccines to employ oneor more preservatives to help prevent contamination by bacteria, fungusor both. Among the most widely employed preservatives have been phenol,2-phenoxyethanol, or thimerosal (which contains mercury). Concerns havebeen expressed as to the potential efficacy, toxicity or mutagenicity ofone or more of the above agents or other ingredients of vaccines. Therehas been a recognized resurgence in vaccine research in recent years,which has placed an even more acute demand upon vaccine manufacturers toaddress the above issues.

Swine Flu Virus is a negative segmented myxovirus. It has infected asignificant portion of the human population. Improved vaccines aredesired. Due to its widespread affect the there is a need for widespreaddistribution of precursors to vaccines and vaccines for this virus. Onechallenge is that many precursors to vaccines and vaccines requirestorage and transport at low temperatures, sub ambient temperatures.Thus it presents a challenge to provide such vaccines to populationslocated in places that do not have infrastructure for storage andtransport of such precursors at sub ambient temperatures. To preparevaccines, viruses are inactivated so that they do not infect the hostand the antigens are stabilized so that hosts inoculated with theinactivated viruses develop immunological responses. The United StatesDepartment of Agriculture has approved protocols for using binaryethylene-imine or formaldehyde to inactivate certain viruses for vaccineproduction. In U.S. Pat. Nos. 5,459,073; 5,811,099; and 5,849,517, thereare disclosed preservative compositions that include a formaldehydedonor agent, and specifically, one or both of diazolidinyl urea orimidazolidinyl urea. That such compositions can be used in a vaccine isaddressed in U.S. Pat. Nos. 5,811,099; and 5,849,517. Commonly owned andcopending U.S. application Ser. No. 12/940,112 filed Nov. 5, 2010 titled“INACTIVATED VIRUS COMPOSITIONS AND METHODS OF PREPARING SUCHCOMPOSITIONS”, now published as US 2011/0110975, discloses contacting avirus with a formaldehyde donor agent having a molecular weight that isgreater than about 50 g/mol and less than about 400 g/mol for a periodof time (e.g., at least about 12 hours) sufficient for de-activating theinfectious component with the formaldehyde donor agent and forpreserving at least a portion of the surface antigens to form adeactivated virus and the resulting composition. This application isincorporated herein by reference.

Notwithstanding the foregoing, there remains a need in the art for saferand less toxic inactivated live virus compositions useful in vaccines,more specifically, veterinary vaccines, and still more specifically,inactivated-viral swine vaccines, which can be produced in high yield,with inconsequential toxicity or other potential undesired side effects.What is further needed are vaccine precursors and vaccines that can bestored and transported at ambient temperatures and processes for theirproduction, transport and storage at ambient temperatures. What isneeded are compositions that can be manufactured in an efficient manner,that contain components which enhance the disinfectant and antimicrobialproperties of the vaccine precursors, that exhibit hemagglutinationequal to or better than industry standards, compositions that do notexhibit a loss in titer and are relatively non-toxic.

SUMMARY OF THE INVENTION

The present invention is directed at a composition comprising a liveswine flu virus having an infectious component and a plurality ofsurface antigens on the virus in contact with a formaldehyde donor agenthaving a molecular weight that is less than about 400 g/mol. Preferably,the formaldehyde donor agent is selected from a non-crosslinkingchemical fixative that contains urea.

The present invention provides a method for deactivating a live swineflu virus having an infectious component and a plurality of surfaceantigens, comprising the steps of: a) providing a live swine flu virushaving an infectious component and a plurality of surface antigens; andb) contacting the virus swine flu with a formaldehyde donor agent havinga molecular weight that is greater than about 50 g/mol and less thanabout 400 g/mol for a period of time (e.g., at least about 12 hours)sufficient for de-activating the infectious component with theformaldehyde donor agent and for preserving at least a portion of thesurface antigens to form a deactivated swine flu virus. In anotherembodiment, the invention is a method of preparing a composition usefulas a vaccine comprising the abovementioned steps in combination with thestep of c) mixing a non-toxic effective amount, for inducing an immuneresponse in a subject to which the vaccine is administered, of thedeactivated swine flu virus with a pharmaceutically acceptable carrier.Preferably the composition containing a pharmaceutically acceptablecarrier is useful in, or as, a vaccine composition.

The present invention provides a method for deactivating a live virushaving an infectious component and a plurality of surface antigens,comprising the steps of: a) providing a live virus having an infectiouscomponent and a plurality of surface antigens; and b) contacting thevirus with a formaldehyde donor agent having a molecular weight that isgreater than about 50 g/mol and less than about 400 g/mol for a periodof time (e.g., at least about 12 hours) sufficient for de-activating theinfectious component with the formaldehyde donor agent and forpreserving at least a portion of the surface antigens to form adeactivated virus; and c) storing and/or transporting the contactedcomposition at ambient temperatures, that is without cold storage ortransport. In another embodiment, the invention is a method of preparinga composition useful as a vaccine comprising the abovementioned steps incombination with the step of d) mixing a non-toxic effective amount, forinducing an immune response in a subject to which the vaccine isadministered, of the deactivated virus with a pharmaceuticallyacceptable carrier. Preferably the composition containing apharmaceutically acceptable carrier is useful in, or as, a vaccinecomposition.

The invention herein also contemplates vaccine compositions preparedaccording to the methods or using the virus of the foregoing aspects ofthe invention. The aspects of the present invention offer a number ofadvantages as compared with existing compositions useful in or asvaccines including the possibility to obtain total protection ofmortality without use of an adjuvant; the ability to withstand withoutdamaging the vaccine and/or without aggregating vaccine components afterone or more freeze-thaw cycles; a reduction of cross-linking surfaceantigens to a significant enough degree that would result in anauto-immune hypersensitivity reaction mediated by mast cells is avoided;the vaccine protects birds from a live virus challenge (e.g., birds aregiven two vaccinations two weeks apart, and hemagglutination inhibition(HAI) data from samples drawn after the first vaccination indicate thata single vaccination is sufficient); a substantial reduction in theamount of antigen (e.g., a reduction by at least two logs in antigen,and more preferably at least three logs in antigen) necessary tomanufacture the vaccine may be realized, a very important economicconsideration. The compositions of the invention or prepared by themethod of the invention facilitate storage and transportation of vaccineprecursors and vaccines at ambient temperatures, that is without theneed for storage and/or transportation with refrigeration. Thecompositions of the invention can be manufactured in an efficientmanner, contain components which enhance the disinfectant andantimicrobial properties of the vaccine precursors, exhibithemagglutination equal to or better than industry standards, do notexhibit a loss in titer and are relatively non-toxic. The invention isuseful with flu antitgens produced in singular or plural forms in anybiological system. Such antigens may be produced in recombinant systems.In particular such antigens may be subjected to the stabilizing andefficacy treatments of the invention.

DESCRIPTION OF FIGURES

FIGS. 1 to 28 show graphs of Bio-Activity of IDU stabilized compositionsover time as described in the Examples.

DETAILED DESCRIPTION

The explanations and illustrations presented herein are intended toacquaint others skilled in the art with the invention, its principles,and its practical application. Those skilled in the art may adapt andapply the invention in its numerous forms, as may be best suited to therequirements of a particular use. Accordingly, the specific embodimentsof the present invention as set forth are not intended as beingexhaustive or limiting of the invention. The scope of the inventionshould, therefore, be determined not with reference to the abovedescription, but should instead be determined with reference to theappended claims, along with the full scope of equivalents to which suchclaims are entitled. The disclosures of all articles and references,including patent applications and publications, are incorporated byreference for all purposes. Other combinations are also possible as willbe gleaned from the following claims, which are also hereby incorporatedby reference into this written description.

The present invention is directed at a composition comprising adeactivated swine flu virus having an infectious component and aplurality of surface antigens in contact with a formaldehyde donor agenthaving a molecular weight that is less than about 400 g/mol. In additionthe present invention provides an improved method for deactivating alive swine flu virus having an infectious component and a plurality ofsurface antigens, comprising the steps of: a) providing a live swine fluvirus having an infectious component and a plurality of surfaceantigens; b) contacting the swine flu virus with a formaldehyde donoragent having a molecular weight that is greater than about 50 g/mol andless than about 400 g/mol for a period of time sufficient forde-activating the infectious component with the formaldehyde donoragent, and for preserving at least a portion of the surface antigens toform a deactivated swine flu virus. In another embodiment the inventionis a method of preparing a composition comprising the abovementionedsteps in combination with the step of c) storing or transporting themixture at ambient temperatures. In another embodiment the invention isa method of preparing a composition comprising the abovementioned stepsin combination with the step of d) mixing a non-toxic effective amount,for inducing an immune response in a subject to which the vaccine isadministered, of the deactivated swine flu virus with a pharmaceuticallyacceptable carrier. Preferably the composition containing apharmaceutically acceptable carrier is useful in, or as, a vaccinecomposition.

In another embodiment, the invention is a method comprising the stepsof: a) providing a live swine flu virus having an infectious componentand a plurality of surface antigens; b) contacting the swine flu, viruswith a formaldehyde donor agent, having a molecular weight that isgreater than about 50 g/mol and less than about 400 g/mol for a periodof time sufficient for de-activating the infectious component with theformaldehyde donor agent, and for preserving at least a portion of thesurface antigens to form a deactivated virus c) transporting or storingthe contacted composition at ambient temperature.

The method for preparing a stabilized live virus composition may furthercomprise any one or more of the features described in this specificationin any combination, including the preferences and examples listed inthis specification, and includes the following features: the virus isgrown in a tissue or in-vitro cell culture, such as a chicken egg; theformaldehyde donor agent is selected from a non-crosslinking chemicalfixative that contains urea; the formaldehyde donor agent is selectedfrom diazolidinyl urea (DU), imidazolidinyl urea (IDU), or a mixturethereof; the contacting step includes contacting the virus with theformaldehyde donor agent having a concentration of less than about w/v(grams per 100 ml total volume) or less; the contacting step (b) occursfor a period of about 24 to about 240 hours and more preferably about 24to about 72 hours; the contacting step (b) occurs at a temperature ofabout 23° C. to about 37° C.; the method is free of any step ofcontacting the virus with binary ethylene-imine, formaldehyde, formatin,phenol, 2-phenoxyethanol, thimerosal, bromo-ethylene-imine, ethylmethane sulfonate, Nitrosoguanidine, fluorouracil, 5-azacytadine, or anycombination thereof; the method includes a step of freeze-drying andre-hydrating the antigen-treated virus; further comprising a step ofperforming an assay of the deactivated virus to confirm that theinfectious component has been de-activated; further comprising; c)mixing a non-toxic effective amount for inducing an immune response in asubject to which the vaccine is administered of the deactivated viruswith a pharmaceutically acceptable carrier for forming a vaccinecomposition; the virus is an avian virus provided in a live titer amountof about 10⁶ to about 10¹² EID50 per milliliter of the resulting vaccinecomposition; further comprising a step of contacting the virus with anadjuvant; the mixing step (c) occurs immediately following thecontacting step (b); the contacted composition is transported or storedat ambient temperature; the contacted composition is transported atambient temperature; and ambient temperature is from about 20° C. toabout 37° C.

The compositions may further comprise any one or more of the featuresdescribed in this specification in any combination, including thepreferences and examples listed in this specification, and includes thefollowing features: the virus manufactured by growing the virus intissue or a chicken egg; the formaldehyde donor agent is selected from anon-crosslinking chemical fixative that contains urea; the formaldehydedonor agent is selected from diazolidinyl urea (DU), imidazolidinyl urea(IDU), or a mixture thereof; the formaldehyde donor agent is present ina concentration of less than about 1 w/v (grams per 100 ml total volume)or less; further comprising a pharmaceutically acceptable carrier ordiluent; and the composition can be transported and stored at ambienttemperatures.

The live virus useful in this invention is any live virus having aninfectious component and a plurality of surface antigens which iscapable of being deactivated for use in vaccines. Preferred live virusesare those which can be incubated in tissue or in vitro cell cultures,such as in chicken eggs. Among preferred viruses are avian flu, swineflu, bovine respiratory systhial virus and the like. In one preferredembodiment wherein the composition is transported and/or stored atambient temperatures a preferred virus is a swine flu or an avian virus.More preferably the avian flu virus is Newcastle's disease. Still morespecifically, the virus is a strain of Newcastle Disease Virus selectedfrom a lentogenic strain, a mesogenic strain, a velogenic strain or anycombination thereof. For example, the virus is a La Sota strain (e.g.,Mass. type) of Newcastle Disease Virus.

The amount of time for the contacting step preferably is sufficient forde-activating the virus (whether by attenuating it, modifying it orkilling it), while sufficiently preserving surface antigens so that whena subject is vaccinated with a vaccine composition including thedeactivated virus, the deactivated virus will induce an immunityresponse (e.g., a response that is measurable by HemagglutinationInhibition Assay, such that the response allows the vaccinated subjectsto survive exposure to a lethal dosage of the live virus). In apreferred embodiment, the contacting occurs for a period of time ofabout 12 hours or greater and more preferably about 24 hours or greater.In a preferred embodiment, the contacting occurs for a period of time ofabout 240 hours or less, more preferably 120 hours or less, even morepreferably about 72 hours or less, and most preferably about 48 hours orless. In a preferred embodiment, the contacting step occurs at atemperature of about 20° C. or greater, more preferably about 23° C. orgreater and most preferably about 25° C. or greater. In a preferredembodiment, the contacting step occurs at a temperature of about 37° C.or less, more preferably about 30° C. or less; and most preferably about25 C or less.

The viral infection that is useful in the teachings herein preferably isa swine flu virus. More specifically, the virus is a strain of Swine FluVirus is selected from those designated H1N1, H1N2, H3N2 or the like.When the virus is used in a composition used as a vaccine it is providedin a sufficient amount for invoking an immunity response in a subject towhich the vaccines herein are administered. By way of example, the swinefluid virus may be provided in a live titer amount of about 10³ to about10¹² titer per milliliter of the resulting vaccine composition and morepreferably about 10⁶ to about 10¹¹ titer per milliliter of the resultingvaccine composition. More specifically, the virus titer may range fromabout 10⁶ to about 10⁸ titer per milliliter of the resulting vaccinecomposition. By way of example, the avian virus may be provided in alive titer amount of about 10⁶ to about 10¹¹ titer per milliliter of theresulting vaccine composition. The formaldehyde donor agent is selectedfrom a non-crosslinking, chemical fixative that contains urea. Morepreferably, the formaldehyde donor agent is selected from diazolidinylurea (DU), imidazolidinyl urea (IDU), or a mixture thereof. Theformaldehyde donor agent may consist essentially of diazolidinyl urea(DU) imidazolidinyl urea (IDU), or a mixture thereof.

In the embodiment wherein the virus is an avian flu, the contacting stepgenerally includes contacting the virus with the formaldehyde donoragent having a concentration of about 5 w/v (grams per 100 ml totalvolume) or less and more preferably about 4 w/v or less. Preferably, theformaldehyde donor agent may be present in a concentration of about 0.5w/v (grams per 100 ml total volume) or greater and more preferably aboutw/v (grams per 100 ml total volume) or greater. In the embodimentwherein the virus is a swine flu virus, the contacting step generallyincludes contacting the virus with the formaldehyde donor agent having aconcentration of about 1 w/v (grams per 100 ml total volume) or less andmore preferably about 0.5 w/v or less. Preferably, the formaldehydedonor agent may be present in a concentration of about 0.0625 w/v (gramsper 100 ml total volume) or greater and more preferably about 0.1 w/v(grams per 100 ml total volume) or greater. The contacting step may bethe only step during which the live virus is contacted with theformaldehyde donor agent.

The mixing step is a step that can be employed for preparing theresulting composition useful as a vaccine. The mixing step may occurimmediately following the contacting step (such that there is nointermediate washing, quenching step or other step by which thedeactivated virus would be subjected to a fluid other than a carrier ofthe vaccine composition), or it may occur during the time period whenthe contacting step (b) is occurring. Thus, it will be appreciated thatduring the mixing step the deactivated virus will remain in contact withsolution that includes the formaldehyde donor agent. During the mixingstep, the antigen-treated virus may be maintained at a temperature aboveabout 15° C. (e.g., it may be about room temperature).

The composition of the invention generally will thus include apharmaceutically acceptable carrier or diluent, which may be a liquid, asolid, a gel or otherwise. By way of illustration, the antigen-treatedvirus may be admixed in a suitable carrier (e.g., one based on a wateror a saline solution) that optionally is buffered (e.g., phosphatebuffered saline, such as Dulbecco's phosphate buffered saline “D-PBS”)before administering into a subject animal. Preferably, the carrier issuch that the antigen-treated virus is uniformly dispersed in theresulting composition at the time of the administration, and it will notdegrade the antigen-treated virus throughout a storage life of at least10 days, more preferably at least one month at a temperature of about 0°C. to about 37° C. An example of one suitable solution includes, amixture of CaCl₂ (Calcium Chloride); MgCl₂ (Magnesium Chloride); KCI(Potassium Chloride); KH₂PO₄ (Potassium Phosphate, monobasic); NaCl(Sodium Chloride); Na₂HPO₄ (Sodium Phosphate, dibasic); and DGlucose(dextrose). More specifically, one example of such a solution is setforth in the following Table 1, where mM refers to milli-molarity;millimoles/liter.

TABLE I CaCl₂ 0.901 mM MgCI₂ 0.493 mM KCl 2.67 mM KH₂PO₄ 1.47 mM NaCI137.93 mM Na₂HPO₄ 8.06 mM D-Glucose 5.56 mM

As taught in U.S. Pat. No. 7,252,984, a carrier or diluent may includeone or any combination of stabilizers, preservatives and buffers.Suitable stabilizers may include, for example SPGA, carbohydrates (suchas sorbitol, mannitol, starch, sucrose, peptone, arginine, dextran,glutamate or glucose), proteins (such as dried milk serum, albumin orcasein) or degradation products thereof. Suitable buffers may includefor example alkali metal phosphates. Suitable preservatives may includethimerosal, merthuilate and gentamicin. Diluents include water, aqueousbuffer (such as buffered saline) and polyols (such as glycerol). It willbe appreciated that vaccine compositions herein, as well as any of itscarrier or diluents is preferably free of any anti-biotic, and/or anymercury-containing ingredient.

The method may further comprise a step of contacting the virus with anadjuvant. Any suitable adjuvant may be utilized in the compositions andmethods of this invention. Exemplary adjuvants include those selectedfrom one or any combination of lecithin, a pharmaceutically acceptablepolymer, saponin or a derivative thereof, cholesterol, aluminiumhydroxide, -phosphate or -oxide, oil-in-water or water-in-oil emulsionbased on, for example a mineral oil, a vegetable oil (such as vitamin Eacetate), or some other suitable carboxylic acid. See, e.g., U.S. Pat.No. 5,152,981, incorporated by reference. If an adjuvant is employed, itmay be employed in a suitable amount for providing adjuvant activity.Thus, by way of example, following treatment of the surface antigens ofthe virus, the deactivated virus may diluted into D-PBS, which mayinclude an adjuvant or be free of an adjuvant. Other examples ofsuitable adjuvants may include one or any combination of compounds orcompositions for this purpose

The composition and method for making it is a relatively simple andelegant to a technology that previously has seen various potentiallycomplicating approaches. Though such approaches may be employed with theteachings herein, it is preferable that the vaccine composition and thevirus are free of any contact with any plant-cell-produced components:the virus includes or is free of an infectious genetically engineered,genetically modified or cloned virus; the method may optionally be freeof any step of contacting the virus with binary ethylene-imine,formaldehyde, formalin, phenol, 2-phenoxyethanol, thimerosal,bromo-ethylene-imine, ethyl methane sulfonate, nitrosoguanidine,fluorouracil, 5-azacytadine, or any combination thereof; or anycombination of the foregoing. It is also possible that the vaccinecomposition may include or be free of any neutralizing antibodies boundto the surface antigens. It is also possible that the vaccinecomposition may otherwise include or be free of an antioxidant.

Other variations of the invention are also possible. To illustrate, themethod may include a step of freeze-drying and re-hydrating theantigen-treated virus. The antigen-treated virus may include or be freeof any temperature reduction to a temperature below about 10° C. (e.g.,to below about 0° C.). The method may include or be free of any stepthat removes the formaldehyde donor agent prior to the mixing step.Accordingly, the various aspects of the invention contemplate that theadministration of the vaccine composition is accomplished in the absenceof any step of diluting the composition at the time of or at the site ofadministration of the vaccination. The mixing step described previouslycould constitute the only diluting step that occurs in the methodsherein. The method may also include one or more steps of storing theresulting composition for a period of at least 30 days, at least 60days, or even at least 90 days following the mixing step; storing theresulting composition at one or more temperatures of below about 1 St(e.g., about 0 to about 10° C.), with the composition retaining itsstability under such conditions (e.g., the amount of useful deactivatedvirus remains within about 20% and more preferably within about 10% ofthe titer amount at the time of the mixing step). The composition may bestored in suitable sealed containers, such as stopped vials (e.g.,rubber stopped glass or plastic vials), sealed syringes, blisterpackaging, or otherwise. Preferably the method includes storing ortransporting any composition of the invention or prepared by the methodsof the invention at ambient temperature, wherein ambient temperature isdefined hereinbefore. When such compositions are stored or transportedat ambient temperatures such compositions retain their efficacy asdescribed hereinbefore.

The method may also include one or more steps of performing an assay ofthe deactivated virus to confirm that the infectious component has beende-activated. The methods herein further contemplate immunizing asubject with a vaccine composition prepared according to the presentteachings. Thus the methods herein may further comprise at least onestep of administering a subject in need of immunization with the vaccinecomposition described in the present teachings. Any step ofadministering a subject in need of immunization with the vaccinecomposition by performed by ingestion (e.g., from drinking water),intranasally (e.g., by aerosol), intraocularly (e.g., by aerosol), viaintramuscular injection, by subcutaneous injection, by delivering intoeggs prior to hatching (e.g., manually or by machine); or anycombination thereof. A plurality of steps of administering the vaccineto the subject may be performed at intervals (e.g., they may beadministered to the same subject on multiple occasions, such as atintervals of at least 3 days). By way of further example, at least twosteps of administering the subject may performed at intervals of greaterthan one week (e.g., about 14 days apart). It is also possible that onlya single dose is administered and is sufficient for achieving thedesired satisfactory immune response.

As gathered from the foregoing, another aspect of the present inventionis directed at deactivated virus described herein that has beencontacted with a formaldehyde donor agent according to the aboveteachings, as well as compositions useful as vaccines (including the andone or more of the other ingredients, e.g., one or more of a carrier, anadjuvant, or both) resulting from the methods herein. Resulting virusestreated according to the teachings herein may be characterized as beingfree of cross-linking with other surface antigens of the virus.

The dosage of the vaccine preferably will be a sufficient amount forinducing immunity in the vaccinated subjects against challenge by avirulent form of the virus, wherein immunity can be described as therealization within a period of 4, days, more preferably 7 days, andstill more preferably 14 days after challenge of a death rate due to thevirulent form of the virus that is less than 50% of the population ofsubjects challenged, more preferably less than 25% of subjectschallenged and still more preferably less than 5% of subjects challenged(e.g., approximately 100% of challenged subjects survive the challenge).Challenges include the step of administering the virulent form of thevirus to the subject in a 100% lethal dosage amount.

Though illustrated in connection with immunization for prevention ofswine flu virus, the present invention is not intended to be so limited.It may have application for immunization for prevention of otherviruses, including but not limited to other avian paramyxoviruses, avianinfluenza (e.g., H5 or H7 influenza, such as H5N1 influenza), avianpolyoma virus, Pacheco's disease, West Nile Virus, diminuvirus, chickenanemia virus, or Circo virus. The vaccine compositions herein may alsoinclude a plurality of antigenic components suitable for immunizingagainst a plurality of viruses. For example, the invention contemplatesthat the resulting vaccine may immunize against Swine Flu Virus and oneor more other viruses. The methods and compositions herein may beemployed in the immunization of mammal and avian subjects.

The method of the invention comprises the steps of: providing a livevirus having an infectious component and a plurality of surfaceantigens; b) contacting the virus with a formaldehyde donor agent havinga molecular weight that is greater than about 50 g/mol and less thanabout 400 g/mol for a period of time sufficient for de-activating theinfectious component with the formaldehyde donor agent, and forpreserving at least a portion of the surface antigens to form adeactivated virus. The method of the invention may further comprise oneor more of the following features in any combination. The virus is grownin a chicken egg; the virus is Swine flu virus; the formaldehyde donoragent is selected from, or consists essentially of, a non-crosslinkingchemical fixative that contains urea. The formaldehyde donor agent isselected from, or consists essentially of, diazolidinyl urea (DU),imidazolidinyl urea (IDU), or a mixture thereof. The resulting solutionconsists essentially of de-activated virus and the formaldehyde donoragent; the contacting step includes contacting the virus with theformaldehyde donor agent having a concentration of less than about 1 w/v(grams per 100 ml total volume) or less, preferably about 0.5 w/v orless (grams per 100 ml total volume) and preferably about 0.625 w/v(grams per 100 ml total volume, or greater), and more preferably aboutof about 0.1 w/v (grams per 100 ml total volume) or greater. Thecontacting step (b) occurs for a period of about 24 to about 72 hours.The contacting step (b) occurs at a temperature of about 20° C. to about37° C., preferably about 25° C. The mixing step (c) occurs immediatelyfollowing the contacting step (b). During the time period throughout thecontacting step (b) the antigen-treated virus is maintained at atemperature of about 20° C. or greater. The contacting step (b) is theonly step during which the live virus is contacted with the formaldehydedonor agent. The resulting composition may be free of any contact withany plant-cell-produced components. The virus includes an infectiousgenetically engineered, genetically modified or cloned virus. The virusmay be free of any infectious genetically engineered, geneticallymodified or cloned virus. The method may be free of any step ofcontacting the virus with binary ethylene-imine, formaldehyde, formalin,phenol, 2-phenoxyethanol, thimerosal, bromo-ethylene-imine, ethylmethane sulfonate, Nitrosoguanidine, fluorouracil, 5-azacytadine, or anycombination thereof. The method may include a step of freeze-drying andre-hydrating the antigen-treated virus. The method may further comprisea step of performing an assay of the deactivated virus to confirm thatthe infectious component has been de-activated. The method may be freeof any step of reducing the temperature of the virus to below about 20°C. The surface antigens may be free of cross-linking with other surfaceantigens of the virus. The method may further comprise; c) mixing anon-toxic effective amount for inducing an immune response in a subjectto which the vaccine is administered of the deactivated virus with apharmaceutically acceptable carrier for forming a vaccine composition.The virus is an avian virus provided in a live titer amount of about 10⁸to about 10¹¹ Eid per milliliter of the resulting vaccine composition.The method may further comprise a step of contacting the virus with anadjuvant. The mixing step (c) may occur immediately following thecontacting step (b); during the time period throughout the contactingstep (b) and the mixing step (c). The antigen-treated virus ismaintained at a temperature of about 20° C. During the time periodthroughout the contacting step (b) and the mixing step (c), theantigen-treated virus may be free of any temperature reduction to atemperature below about 20° C. The method may be free of any step thatremoves the formaldehyde donor agent prior to the mixing step (c). Thecomposition of the invention comprises a deactivated virus having aninfectious component and a plurality of surface antigens in contact witha formaldehyde donor agent having a molecular weight that is less thanabout 400 g/mol. The composition of the invention may further compriseone or more of the following features in any combination. Theformaldehyde donor agent is selected from, consists essentially of, anon-crosslinking chemical fixative that contains urea. The formaldehydedonor agent is selected from, consists essentially of, diazolidinyl urea(DU), imidazolidinyl urea (IDU), or a mixture thereof. The formaldehydedonor agent is present in a concentration of about 1 w/v (grams per 100ml total volume) or less and more preferably about 0.5 w/v (grams per100 ml total volume) or less and about 0.625 w/v (grams per 100 ml totalvolume) or greater and more preferably about 0.1 (grams per 100 ml totalvolume) or greater. The composition further comprises a pharmaceuticallyacceptable carrier or diluent. The resulting composition may be free ofany contact with any plant-cell-produced components. The virus mayinclude an infectious genetically engineered, genetically modified orcloned virus; the virus is free of any infectious geneticallyengineered, genetically modified or cloned virus. The composition may befree of binary ethylene-imine, formaldehyde, formalin, phenol,2-phenoxyethanol, thimerosal, bromo-ethylene-imine, ethyl methanesulfonate, Nitrosoguanidine, fluorouracil, 5-azacytadine, or anycombination thereof. The surface antigens may be free of cross-linkingwith other surface antigens of the virus. A non-toxic effective amountfor inducing an immune response in a subject to which the vaccine of thedeactivated virus of a pharmaceutically acceptable carrier isadministered. The virus may be an avian virus provided in a live titeramount of about 10⁶ to about 10¹¹ EID₅₀ per milliliter. The compositionretains its immunological efficacy after storage and transportation atambient temperature, about 20° C. to about 37° C. and more preferablyabout 23° C. to about 30° C.

Illustrative Embodiments of the Invention

The following examples are provided to illustrate the invention, but arenot intended to limit the scope thereof. All parts and percentages areby weight unless otherwise indicated.

Test Methods

Inactivation Sample Hemagglutination Assay (HA) is run to determine ifinactivation treatment degrades virus surface epitopes necessary forhemagglutination. If so, the HA result should be negative. The desiredresult is positive hemagglutination, thereby confirming that surfaceepitopes are preserved. More specifically, in this assay, a sample isincubated with a fresh preparation of chicken red blood cells (cRBC5).Hemagglutination (HA) is defined as the clumping of cRBCs. The virus canpromote hemagglutination through the interaction of molecules on thevirus surface with molecules on the surface of cRBC5. Damaged ordegraded virus does not promote hemagglutination. Depending on thesample used (treated/untreated virus or allantoic fluid—AF—frominoculated eggs), the results may be interpreted differently. If thesample is treated/untreated virus (the inactivation sample) a positiveHA result indicates that the virus is not degraded due to thetreatment—it gives no information (in this case) about the viability ofthe virus (inactivation state). If the sample is the allantoic fluidfrom an inoculated egg, a positive HA result indicates that the virus isnot inactivated by the treatment (the virus was active and able toreplicate in the egg). If the virus is completely inactivated by thetreatment (eg—incubation with DU/IDU/BEI/formaldehyde), the small amountof virus in the inoculum would be diluted out by the large volume of AF,and the HA result would be negative.

Embryonic Toxicity assay is run to determine if the virus is stillviable after the inactivation treatment. If still viable the virus wouldreplicate in allantoic fluid and kill an embryo to which it iscontacted. The desired result is embryonic viability, thereby confirmingthe presence of no live virus. More specifically, embryonic toxicity isassessed by candling the eggs, such that an egg is placed on a source ofbright light (e.g. a flashlight), and the embryo is directly observed.Viable embryos are identified by certain visual cues (pulsatilevasculature, for example). In these experiments, embryonic death may bedue to one of three things: (1) injury/infection resulting from theneedle stick used in the injection; (2) chemical toxicity (byinactivating agent—DU/IDU); or (3) live virus replication (as a resultof incomplete NDV inactivation).

Allantoic fluid (AF; from injected eggs) HA Assay is run to determine ifthe virus is not viable. If not viable, inactivated virus would notreplicate and injected virus would be diluted out in AF. If, after‘inactivation’ treatment, the virus is still viable, allantoic fluidwould contain high enough titer of virus to promote hemagglutination.The desired result is negative hemagglutination, thereby confirming thatthe AF fluid contains no live virus.

Chicken Embryonic Fibroblast (CEF) Immunofluorescence (IF) Assay is runto determine whether successful inactivation occurs. It is recognizedthat chicken embryonic fibroblasts (CEF) grow well in culture and arehighly susceptible to infection by live Swine Flu. In this assay,allantoic fluid (AF) from inoculated eggs is applied to confluent CEFmonolayers. After 5 days, cells are washed, fixed and stained withanti-viral, antibodies. Positive staining indicates that there was livevirus in AF, and therefore, ‘inactivation’ treatment is unsuccessful.The desired result is no staining (which indicates that the virus hadbeen completely inactivated), and supports that the AF fluid contains nolive virus.

Hemagglutination Inhibition Assay is run with serum samples obtainedfrom vaccinated and/or control animals are incubated with live virusprior to use in the aforementioned HA assay. If the cRBCs do notagglutinate (negative result), the conclusion is that there werespecific anti-viral antibodies in the serum (seroconversion) whichblocked the viral antigens from interacting with the cRBCs. A serum HA1titer of greater than 16 correlates with an animal successfullyrejecting a subsequent live viral challenge. An example of one suchassay is found in Allan and Gough, “A standard haemagglutinationinhibition test for Newcastle disease.(2) Vaccination and Challenge”.Vet Rec., Vol. 95, Issue 7, 147-149, Aug. 17, 1974.

Test Results

Testing is performed on Swine influenza virus (SIV) H1N1. The virus ischaracterized in red blood cells (RBC) a permissive cell type forassaying the hemagglutination (HA) activity and cytopathic effect (CPE)is identified. Imidazolidinyl urea preparations are prepared inDulbecco's phosphate buffered saline at concentrations as describedhereinafter. Equal volumes of virus are mixed with the imidazolidinylurea. The resulting solutions are then serially diluted and added tocell monolayers. The plates are incubated at 37° C., in 5% carbondioxide for 5 to 6 days. The monolayers are observed for morphologicalchanges associated with cytotoxicity effect (CTE) or CPE caused byimidazolidinyl urea and Swine Influenza Virus.

Concentrations of 0.0625, 0.1250, 0.250 and 0.500 of imidazolidinyl urea(IDU) are tested according to the protocol described. The results arecompiled in Table 2.

TABLE 2 HA CTE CPE Titer Titer Titer CTE per ml above IDU per ml per mlTiter (above CTE Conc SIV SIV per ml titer) SIV Example % PresentPresent No SIV Present 1 0.5000 1920 1280 1280 40 2 0.2500 1920 12801280 0 3 0.1250 1920 640 640 0 4 0.0625 1920 160 160 80 5 0 1280 40960

The data demonstrates that imidazolidinyl urea levels of 0.0625, 01250,0.2500 and 0.5000 levels significantly reduce SIV infectivity withincubations of 37° C. for 18 hours.

The inactivation of SIV infectivity using imidazolidinyl urea, BEI andan untreated SIV control is studied. The virus is prepared as describedhereinbefore. The virus is expanded to a total of 350 ml and theinfectivity and HA bioactivity is determined as approximately 8.00TCID₅₀/ml and 1280 HA/ml. Infectivity is determined using a MDCK cellline and microscopic observations for cytopathic effects as the 50percent end point. Complete inactivation is tested using fluorescentantibody to type A influenza. The HA bioassay is performed using chickenred blood cells that are observed and scored for hemaglutination. Threetreatment groups are prepared, IDU treated, BEI treated and untreated.Each preparation is incubated at room temperature for approximately 72hours with continuous rocking. After incubation, infectivity and HAbioactivity are tested using MDCK and chicken red blood cells,respectively. Each preparation is aloquoted into four volumes and placedat 2-7° C., 22-25° C., 35-38° C., and 40-42° C. Samples are collected at0 days, 7 days, 14 days, 21 days, 1 month, 3 months, 6 months and 12months. the HA bioactivity is tested on the day of collection at eachtime point. A 3 percent BEI solution is prepared and 45 ml is mixed with150 ml of virus lysate. A 10 percent stock of imidazolidinyl urea isprepared in sterile RO/DI water and filter stabilized. A 0.5 percentimidazolidinyl urea concentration a 7.5 ml portion of the 1.0 percentstock is mixed with 150 ml of virus lysate. Infectious titers of thebase samples are determined for the untreated version as 8.00 TCID₅₀/mland 8.50 FAID₅₀/ml. For BEI treated and IDU treated samples, the resultsare below the level of detection, where the Level of detection is ≥1.3TCID₅₀/ml and 1.3 FAID₅₀/ml.

The results of the Hernagglutination bio-activity is compiled in Table3.

TABLE 3 Time Time Time Time Time Time Time Time Time Sample T Days DaysDays Days Months Months Months Months Months type ° C. 0 7 14 21 1 3 3.56 12 SIV 2-7 128 128 256 128 128 128 128 64 256 SIV 22-25 128 256 128128 128 128 64 128 SIV 35-38 128 256 128 128 4 4 <2 16 SIV 40-42 128 12832 16 2 2 ND ND BEI 2-7 256 128 256 128 128 128 128 64 128 BEI 22-25 128256 128 128 128 128 16 32 BEI 35-38 128 128 64 64 64 64 2 4 BEI 40-42128 64 4 <2 2 2 ND ND IDU 2-7 256 128 256 128 128 128 128 64 64 IDU22-25 128 256 128 128 128 128 64 4 IDU 35-38 256 256 128 128 32 16 <2 NDIDU 40-42 128 256 128 32 2 2 ND 64 SIV¹ 256 256 256 128 128 128 128 128128 ¹No incubation

Inactivation Studies

H1N1 virus was inactivated using 0.5% DU or 3% v/v BEI at roomtemperature for 72 hours. Preparations of each sample were placed at2-7° C., 22-25° C., 35-39° C. and 40-42° C. for the forced degradationassay. Samples were collected and tested for HA bio-activity every weekfor the first month, at three months, three and a half months, sixmonths, twelve months, fifteen months, eighteen months and twenty-onemonths. After three and a half months the testing of all samples beingstored at 40-42° C. was discontinued due to loss of detectable HAbio-activity. After twelve months, the testing of all samples stored at35-39° C. was discontinued due to loss of detectable HA bio-activity.

Additional HA bio-activity has been tested on the H1N1 isolate stored at2-7° C. and 22-25° C. at fifteen months, eighteen months and twenty-onemonths. All samples collected to date have been tested for HAbio-activity using cRBC. The positive control viruses (H1N1 or H1N2isolates, not-inactivated) had HA titers of 6-8 (log base 2) during thetwenty-one months. The HA bio-activity was retained in all samplestested following inactivation. The HA bio-activity of samples stored at2-7° C. fluctuated between titers of 7 and 8 during the twenty-onemonths of incubation. The only exception was month six in which allsamples had a titer of 6. The results are graphed in FIG. 1. The HAbio-activity of the untreated and IDU-inactivated viruses stored at22-25° C. fluctuated between titers of 6-8 through the fifteen months ofincubation, then decreased to a titer of 4 after twenty-one months ofincubation. The HA bio-activity of the BEI-inactivated virus stored at22-25° C. fluctuated between titers of 7 and 8 through the first threeand a half months of incubation, then was undetectable (titer of <2)after twenty-one months of incubation. The results are graphed in FIG.2. In the first phase of testing, there was no real difference in HAbio-activity between any of the H1N1 isolate treatments stored at 2-7°C. for up to twenty-one months following inactivation. The H1N1 isolateinactivated with IDU demonstrated at least a retention of 3 to 4-foldgreater amount of HA bio-activity compared to the BEI inactivated virusfrom fifteen months through twenty-one months of incubation at 22-25° C.The HA bio-activity of all Phase I samples collected during the firstfifteen months of incubation were tested using one lot of cRBC. The HAbio-activity titers for all samples were within 2 logs of the originaltiter obtained on the day of collection. The only exception is theuntreated virus stored at 40-42° C. at three and three and a half monthswhich had titers that were 3 and 4 logs higher, respectively, than thoseobtained on the original days of collection (see Table 4).

Phase 2 Testing

The purpose of Phase II of this project was to extend the HAbio-activity testing of the H1N2 virus and H3N2 virus from three monthsto one year. The H1N2 and H3N2 viruses were inactivated using IDU andBEI. The H1N2 and H3N2 viruses were inactivated with 0.5% IDU and 3% v/vBEI at room temperature for 72 hours. The un-treated virus samples wereretained at 2-7° C. until the initiation of the forced degradationassay. Preparations of each sample were placed at 2-7° C., 22-25° C.,35-39° C. and 40-42° C. for the forced degradation assay. Samples werecollected and tested for HA bio-activity on days three, seven, ten,fourteen, and twenty-one; one month, two months, three months, fourmonths, six months, nine months and twelve months. After the four monthtesting date all samples being incubated at 40-42° C. were no longertested due to a loss of detectable HA bio-activity.

Treatment of the H1N2 and H3N2 SIV isolates with 0.5% IDU and 3% v/v BEIfor 72 hours resulted in complete SIV infection inactivation (≤1.5TCID₅₀/ml). No cytopathic effect was observed in indicator cells. Asexpected, there was a minimal cytotoxic effect due to the IDU. The H1N2SIV virus yielded a titer of 5.8 TCID₅₀/ml before inactivation and theH1N2 positive control virus yielded a titer of 6.8 TCID₅₀/ml. The H3N2SIV virus yielded a titer of 4.8 TCID₅₀/ml before inactivation and theH3N2 positive control virus yielded a titer of 6.0 TCID₅₀/ml.

The HA bio-activity was tested on days three, seven, ten, fourteen, andtwenty-one; one month, two months, three months, four months, sixmonths, nine months and twelve months. All samples collected to datewere tested for HA bio-activity using cRBC. The H1N2 positive controlhas had an HA titer of 6-7 and the H3N2 positive control has had an HAtiter of 5-7 during the twelve months. The HA bio-activity was retainedin all samples tested following inactivation. The HA bio-activity of theuntreated and BEI-inactivated H1N2 viruses stored at 2-7° C. fluctuatedbetween titers of 6 and 7 through twelve months of incubation. The HAbio-activity of the IDU-inactivated H1N2 viruses stored at 2-7° C.fluctuated between titers of 6 and 7 through the first two months ofincubation then decreased to a titer 4 after twelve months, see FIG. 3.The HA bio-activity of the untreated and BEI-inactivated H1N2 virusesstored at 22-25° C. fluctuated between titers of 6 and 7 through twelvemonths of incubation. The HA bio-activity of the IDU-inactivated H1N2viruses stored at 22-25° C. retained a titer of 6 through the firsttwenty-one days of incubation, then decreased to a titer of 2 aftertwelve months see FIG. 4. The HA bio-activity of the untreated andBEI-inactivated H1N2 viruses stored at 35-39° C. fluctuated, betweentiters of 6 and 7 through the first month of incubation, then wasundetected (titer of <2) after three months. The HA bio-activity of theIDU-inactivated H1N2 viruses stored at 35-39° C. retained a titer of 6for the first three days, retained a titer of 4 through fourteen daysand then gradually decreased below the level of detection to a titer of<2 after three months. The titer for the untreated virus increased to atiter of 1 after nine months, then below the level of detection to atiter of <2 at twelve months. The titer for the IDU-inactivated virusincreased during the six to twelve month testing period see FIG. 5. TheHA bio-activity of the untreated and BEI-inactivated H1N2 viruses storedat 40-42° C. retained a titer of 6 for the first three days ofincubation. The HA bio-activity of the BEI inactivated H1N2 virusdecreased below the level of detection after fourteen days, then had alow level of activity at one month. The HA bio-activity of the untreatedand IDU inactivated H1N2 viruses decreased below the level of detectionafter 1 month, see FIG. 6.

The H1N2 virus inactivated with BEI maintained similar HA bio-activityas the untreated H1N2 virus at the 2-7° C., 22-25° C. and 35-39° C.storage temperatures. The HA bio-activity of the BEI inactivated H1N2virus decreased more rapidly than the untreated virus at 40-42° C. TheHA bio-activity of the H1N2 virus inactivated with IDU decreased at agreater rate at every storage temperature than the untreated or BEIinactivated viruses. The HA bio-activity of the untreated andBEI-inactivated H3N2 viruses stored at 2-7° C. fluctuated between titersof 6 and 7 through twelve months of incubation. The HA bio-activity ofthe IDU-inactivated H3N2 viruses stored at 2-7° C. fluctuated betweentiters of 6 and 7 through the first two months of incubation thenbetween titers of 5 and 6 through twelve months, see FIG. 7. The. HAbio-activity of the untreated and BEI-inactivated H3N2 viruses stored at22-25° C. fluctuated between titers of 6-and 7 through twelve months ofincubation. The HA bio-activity of the IDU-inactivated H3N2 virusesstored at 22-25° C. fluctuated between titers of 6 and 7 through thefirst two months Of incubation, then between titers 5 and 7 throughtwelve months, see FIG. 8. The HA bio-activity of the untreated H3N2virus stored at 35-39° C. fluctuated between titers of 5 and 6 throughthe first four months of incubation, then decreased to a titer of 4after twelve months. The HA bio-activity of the BEI-inactivated and theIDU-inactivated H3N2 viruses stored at 35-39° C. fluctuated betweentiters of 5 and 7 for the first two months, then between titers of 3 and6 through nine months of incubation. After twelve months of incubationthe BEI-inactivated H3N2 virus retained a titer of 3, whereas theIDU-inactivated H3N2 virus HA bio-activity was not detected (titer <2),see FIG. 9. The HA bio-activity of the BEI-inactivated H3N2 virus storedat 40-42° C. was below the level of detection (titer of <2) after sevendays and fluctuated between a below the level of detection and 2 throughfour months. The HA bio-activity of the untreated H3N2 virus fluctuatedbetween a titer of 2 or 3 throughout one month, then was not detectedtwo months. The HA bio-activity of the IDU-inactivated H3N2 virusstarted with a titer of 6 and retained a titer of 5 through the firstmonth of incubation. After two months the IDU-inactivated H3N2 virus hada titer of 3, and then was undetectable after four months. Testing ofall samples stored at 40-42° C. was ceased after four months, see FIG.10.

There was no real difference in the HA bio-activity for any of the H3N2virus treatments through twelve months of storage at 2-7° C., 22-25° C.and 35-39° C.; the only exception being the loss of HA bio-activity tobelow the level of detection of the virus inactivated with IDU on thetwelve month testing date. The HA bio-activity of the H3N2 virusinactivated with IDU appeared to be much more stable than the untreatedor BEI inactivated H3N2 viruses through two months of storage at 40-42°C.

Testing was performed to determine whether the hemagglutinin proteinfrom Swine Influenza Virus (SIV) isolates H1N2 and H3N2 is stabilized bythe compound IDU using expanded inactivation conditions. Data from PhaseI.B. demonstrated that 0.5% IDU was effective in the completeinactivation of SIV infectivity of both isolates (H1N2 and H3N2) after a72 hour incubation at room temperature and did not affect the HAprotein's ability to hemagglutinate red blood cells. However, theincubation of inactivated viruses at elevated temperatures resulted indifferent levels of HA bio-activity stability over time. The level of HAbio-activity stability was not the same for all the isolates tested.

Phase 3 Inactivation Studies Hemagglutination Bio-Activity Testing ofthe H3N2 Isolate Forced Degradation Samples Inactivated with 0.5% IDU

Treatment of all Phase 3 samples with 0.5%, 1.0%, or 2.0% IDU for 24,48, and 72 hours resulted in complete SIV infection inactivation (≤1.5TCID₅₀/ml). No cytopathic effect was observed in indicator cells. Asexpected, there was a minimal cytotoxic effect due to the IDU. The SIVpositive control (not inactivated) yielded a titer of 5.5 TCID₅₀/ml onthe indicator cells.

The H3N2 SIV isolate was inactivated using 0.5% IDU at room temperature,35-39° C. and 40-42° C. for 24, 48 and 72 hours. The H1N2 SIV isolatewas inactivated using 0.5%, 1.0% and 2.0% IDU at room temperature,35-39° C. and 40-42° C. for. 24, 48 and 72 hours. Bulk preparations ofeach sample were placed at 2-7° C., 35-39° C. and 40-42° C. for theforced degradation assay. Samples were collected and tested for HAbio-activity on days 0, seven, fourteen, and twenty-one, and oncemonthly for six months.

The HA bio-activity was tested at tune 0, day seven, day fourteen, daytwenty-one, and once monthly for six months. All samples collected todate have been tested for HA bio-activity using cRBC. The positivecontrol (H3N2 not-inactivated) had an HA bio-activity titer of 6 or 7during the six months. The initial HA bio-activity was retained in allsamples tested regardless of the incubation temperature or time ofincubation used to inactivate the samples. The HA bio-activity of allH3N2 viruses stored at 2-7° C. retained an HA titer of 6 during thefirst month of incubation, then fluctuated between titers of 5 and 6through six months, see FIG. 11. The HA bio-activity of all H3N2 virusesstored at 35-39° C. retained an HA titer of 6 during the first month ofincubation, then fluctuated between titers of 4 and 5 through six monthssee FIG. 12. The HA bio-activity of all H3N2 viruses stored at 40-42° C.retained a titer of 6 for the first seven days then decreased to a titerof 3 after two months. The HA bio-activity of all H3N2 virusesfluctuated between titers of 2 and 4 through five months of incubation,then was not detected (titer <2) after six months, see FIG. 13.

There was no real loss of HA bio-activity for any of the H3N2 virustreatments stored at 2-7° C. through six months. All treatments of theH3N2 virus stored at 35-39° C. retained a HA bio-activity titer of 6through one month, then dropped to a titer of 4 after six months. Alltreatments of the H3N2 virus stored at 40-42° C. retained HAbio-activity titers within 2-fold of each other throughout six monthsexcept at the four month testing date. After four months of storage at40-42° C., there was a 3-fold difference in titer between the virusinactivated at 37° C. for 24 and 48 hours and the virus inactivated at41° C. for 72 hours. However, all treatments stored at 40-42° C.returned to a titer of 3 after five months, then the HA bio-activity wasnot detected after six months.

Phase 4 Hemagglutination Bio-Activity Testing of the H1N2 Isolate ForcedDegradation Samples Inactivated with 0.5%, 1.0% and 2.0% IDU

The HA bio-activity has been tested at time 0, days seven, fourteentwenty-one, and each month for six months. All samples collected to datehave been tested for HA bio-activity using cRBC. The HA titer for thepositive control (H1N2 not-inactivated) remained consistent at 7 or 8during the six months. The incubation temperature, time of inculpation,or concentration of IDU used to inactivate the samples did not affectthe initial HA titer. The HA bio-activity of all H1N2 virusesinactivated with 0.5% IDU and stored at 2-7 ° C. fluctuated betweentiters of 6-8 through six months of incubation, see FIG. 14. The HAbio-activity of all H1N2 viruses inactivated with 0.5% IDU and stored at35-39° C. fluctuated between titers of 7 and 8 through the first sevendays, then fluctuated between titers, of 5 and 7 through four months ofincubation. The HA bio-activity of the H1N2 viruses fluctuated betweentiters of 4 and 5 after five months of incubation and then decreased toa titer of 4 after six months, see FIG. 15. The HA bio-activity of allH1N2 viruses inactivated with 0.5% IDU and stored at 40-42° C.fluctuated between a titer of 7 and 8 for the first seven days, thenfluctuated between a titer of 5 and 6 through three months ofincubation. After six months of incubation the HA bio-activity of allH1N2 viruses decreased to a titer of 2 or 3 see FIG. 16. The HAbio-activity of all H1N2 viruses inactivated with 1.0% IDU and stored at2-7° C. started at a titer of 7 or 8 and fluctuated between titers of 6and 7 through five months of incubation. After six months of incubationthe HA bio-activity of all H1N2 viruses decreased to a titer of 6, seeFIG. 17. The HA bio-activity of all H1N2 viruses inactivated with 1.0%IDU and stored at 35-39° C. started at titers of 7 or 8, then fluctuatedbetween a titer of 5 and 7 through three months of incubation. After sixmonths of incubation the HA bio-activity of all H1N2 viruses decreasedto a titer of 4, see FIG. 18. The HA bio-activity of all H1N2 virusesinactivated with 1.0% IDU and stored at 40-42° C. started at titers of 7or 8, then fluctuated between titers of 4 and 6 through three months ofincubation. After six months of incubation the HA bio-activity of allH1N2 viruses inactivated at room temperature for 24 or 48 hoursdecreased to a titer of 1 and all remaining H1N2 viruses decreased to atiter of 2, see FIG. 19. The HA bio-activity of all H1N2 virusesinactivated with 2.0% IDU and stored at 2-7° C. fluctuated betweentiters of 5-7 through six months of incubation, see FIG. 20. The HAbio-activity of all H1N2 viruses inactivated with 2.0% IDU and stored at35-39° C. started at a titer of 6 or 7, then fluctuated between titersof 4 or 6 through four months of incubation. After six months ofincubation the HA bio-activity of all H1N2 viruses decreased to a titerof 3, see FIG. 21. The HA bio-activity of all H1N2 viruses inactivatedwith 2.0% IDU and stored at 40-42° C. decreased from a titer of 6 or 7to titers of 3 and 4 after two months of incubation. The HA bio-activityof all. H1N2 viruses returned to a titer of 5 after three months. Aftersix months of incubation, the H1N2 viruses inactivated at roomtemperature (24, 48 or 72 hours) or at 37° C. for 24 hours decreasedbelow the level of detection (titer of <2); the H1N2 viruses inactivatedat 37° C. for 48 or 72 hours decreased to a titer of 1; and the H1N2viruses inactivated at 41° C. (24, 48 or 72 hours) decreased to a titerof 2, see FIG. 22.

The H1N2 virus was inactivated using 0.5%, 1.0% and 2.0% IDU at varioustemperatures and incubation periods. Each inactivated preparation wasplaced at 2-7° C., 35-39° C. and 40-42° C. for storage for six months.The initial HA bio-activity of all H1N2 viruses inactivated with 0.5%and 1.0% IDU was slightly higher than the initial HA bio-activity forthe H1N2 viruses inactivated with 2.0% IDU, average of 7.3 and 6.7,respectively. There did not appear to be an overt difference between theinactivation conditions regarding the stability of the HA bio-activityduring incubations at the lower temperatures. However, there was aslight increase in the retention of HA bio-activity in the groups whenincubations were performed at higher temperature. There was no real lossof HA bio-activity for any of the H1N2 viruses stored at 2-7° C.; thetiter for all viruses after six months was within 2-fold of the originaltiter. The pattern of HA bio-activity for all H1N2 viruses stored at35-39° C. was similar; all viruses decreased 3 to 4-fold in titer aftersix months of storage. The pattern of HA bio-activity for all H1N2viruses stored at 40-42° C. was also similar, but the stability of theHA bio-activity appeared to decrease slightly as the concentration ofIDU used for inactivation increased.

All H1N1 isolate samples collected within the first fifteen months ofstorage were also tested using one lot of cRBCs. This testing wasconducted to determine whether differences in HA titers were observedbetween each of the original testing dates (in which various lots ofcRBCs were used) and using one lot of cells. Small differences wereobserved between the titers (within 2-fold) and are probablyattributable to the natural variation of the assay. Samples of theuntreated H1N1 virus collected at 3 and 3.5 months of storage at 40-42°C. had no detectable titer when originally tested (titer <2), but hadtiters of 8 and 16, respectively, when tested using one lot of cRBCs(Appendix II). It is possible that there was some degradation of virionsin these samples that may have caused the number of HA subunits perparticle to increase if all HA subunits disassociated from the virion.The H1N2 and H3N2 isolates were assayed through one year of storage at2-7° C., 22-25° C., 35-39° C. and 40-42° C. and the stability of theIDU-treated samples compared to BEI-treated or untreated samples. Theisolates were inactivated using IDU or BEI at room temperature for 72hours. IDU did not appear to provide much stability of HA bio-activityof the H1N2 preparation used in this phase of the study. The IDU-treatedH1N2 isolates had a lower amount of HA bio-activity at every storagetemperature throughout the 12 month period (FIGS. 3-6). This was nottrue for the H3N2 isolate. Although there was no real difference in HAbio-activity between any of the H3N2 isolate treatments at the 2-7° C.,22-25° C. or 35-59° C. storage temperatures, the IDU-treated isolatemaintained a 3 to 5-fold higher HA bio-activity titer than theBEI-treated isolate at the 40-42° C. storage temperature (FIGS. 7-10).Therefore, it appears that the H3N2 isolate benefitted from treatmentwith IDU if stored at 40-42° C. The H1N2 and H3N2 isolates were alsoassayed through six months of storage at 2-7° C., 35-39° C. and 40-42°C.; however these samples were not compared to BEI or untreatedisolates. Instead, various conditions of IDU-inactivation were compared.No real difference was observed between the HA bio-activity of theisolates inactivated using the various conditions; all conditions testedwere within 2-fold of each other at each testing period (FIGS. 11-22).The H3N2 isolate behaved very similarly between this phase of the study(FIGS. 11-13) and the phase in which the isolate was inactivated only atroom temperature for 72 hours (FIGS. 7-10). The HA bio-activity of theH1N2 isolates inactivated using 0.5% IDU appeared to be slightly morestable than the isolates inactivated with 1.0% or 2.0% IDU at theelevated temperatures (35-39° C. and 40-42° C.). This supports previousstudies that suggested a range of 0.25% to 0.5% IDU was useful toinactivate virus and stabilize HA bio-activity. The H1N2 preparationused in this phase of the study (FIGS. 14-22) appeared to be much morestable than the preparation used in the earlier studies where theisolate was inactivated at room temperature for 72 hours (FIGS. 3-6);this was most evident especially at the higher temperatures. This may bebecause the H1N2 isolate used in this phase of the study was re-expandedand the new material had a 2-fold higher HA bio-activity titer than theprevious material. The increased number of virions and the resultingchange in the ratio of IDU to virus mass may have aided in its overallgreater stability in this phase of the study. These results suggest thatIDU is beneficial for stabilizing the HA bio-activity of the H1N1 andH3N2 isolates, compared to BEI-treatment, when stored at elevatedtemperatures. The H1N2 isolate also appeared to have benefitted fromtreatment with IDU, but not this was not consistent between twopreparations of the virus. There is a greater amount of HA stabilityobserved with the H1N2 isolate if the initial HA bio-activity is higher(at least 7), but the stabilization of this isolate at the higherinitial titer was not compared to BEI-treatment. These results alsosupport previous data suggesting that a concentration of 0.5% IDU isoptimal for use in inactivating virus and stabilizing HA bio-activity.

Table 4 shows the titer data measured.

FIGS. 23 to 25 provide a comparison of the HA Bio-Activity for the H3N2Virus Inactivated with 0.5% IDU for 72 Hours at Room Temperature betweenPhase I and Phase II. FIGS. 26 to 28 provide a comparison of the HABio-Activity for the H1N2 Virus Inactivated with 0.5% IDU for 72 Hoursat Room Temperature between Phase I and Phase II

Parts by weight as used herein refers to 100 parts by weight of thecomposition specifically referred to.

Any numerical values recited in the above application include all valuesfrom the lower value to the upper value in increments of one unitprovided that there is a separation of at least 2 units between anylower value and any higher value. As an example, if it is stated thatthe amount of a component or a value of a process variable such as, forexample, temperature, pressure, time and the like is, for example, from1 to 90, preferably from 20 to 80, more preferably from 30 to 70, it isintended that values such as 15 to 85, 22 to 68, 43 to 51, 30 to 32 etc.are expressly enumerated in this specification. For values which areless than one, one unit is considered to be 0.0001, 0.001, 0.01 or 0.1as appropriate. These are only examples of what is specifically intendedand all possible combinations of numerical values between the lowestvalue and the highest value enumerated are to be considered to beexpressly stated in this application in a similar manner. Unlessotherwise stated, all ranges include both endpoints and all numbersbetween the endpoints. The use of “about” or “approximately” inconnection with a range applies to both ends of the range. Thus, “about20 to 30” is intended to cover “about 20 to about 30”, inclusive of atleast the specified endpoints. The term “consisting essentially of” todescribe a combination shall include the elements, ingredients,components or steps identified, and such other elements ingredients;components or steps that do not materially affect the basic and novelcharacteristics of the combination. The, use of the terms “comprising”or “including” to describe combinations of elements, ingredients,components or steps herein also contemplates embodiments that consistessentially of the elements, ingredients, components or steps. Plural,elements, ingredients, components or steps can be provided by a singleintegrated element, ingredient, component or step. Alternatively, asingle integrated element, ingredient, component or step might bedivided into separate plural elements, ingredients, components or steps.The disclosure of “a” or “one” to describe an element, ingredient,component or step is not intended to foreclose additional elements,ingredients, components or steps.

What is claimed is:
 1. A method comprising the steps of: a) providing alive swine flu virus having an infectious component and a plurality ofsurface antigens; b) contacting the swine flu virus with a formaldehydedonor agent haying a molecular weight that is greater than about 50g/mol and less than about 400 g/mol for a period of time sufficient forde-activating the infectious component with the formaldehyde donoragent, and for preserving at least a portion of the surface antigens toform a deactivated virus.
 2. A method of claim 1 wherein the virus isgrown in a tissue or in vitro cell culture.
 3. A method of claim 1 or 2wherein the formaldehyde donor agent is selected from a non-crosslinkingchemical fixative that contains urea.
 4. A method of claim 3 wherein theformaldehyde donor agent is selected from diazolidinyl urea (DU),imidazolidinyl urea (IDU), or a mixture thereof.
 5. A method of any oneof claims 1 to 4 wherein the contacting step includes contacting thevirus with the formaldehyde donor agent having a concentration of lessthan-about w/v (grams per 100 ml total volume) or less.
 6. A method ofany one of claims 1 to 5 wherein the contacting step (b) occurs for aperiod of about 24 to about 72 hours.
 7. A method of any one of claims 1to 6 wherein the contacting step (b) occurs at a temperature of about23° C. to about 37° C.
 8. A method of any one of claims 1 to 7 whereinthe method is free of any step of contacting the virus with binaryethylene-imine, formaldehyde, formalin, phenol, 2-phenoxyethanol,thimerosal, bromo-ethylene-imine, ethyl methane sulfonate,Nitrosoguanidine, fluorouracil, 5-azacytadine, or any combinationthereof.
 9. A method of any one of claims 1 to 8 wherein the methodincludes a step of freeze-drying and re-hydrating the antigen-treatedvirus.
 10. A method of preparing a vaccine comprising the method of anyone of claims 1 to 9 which further comprises; c) mixing a non-toxiceffective amount for inducing an immune response in a subject to whichthe vaccine is administered of the deactivated virus with apharmaceutically acceptable carrier for forming a vaccine composition.11. A method of claim 10 wherein the virus is an avian virus provided ina live titer amount of about 10⁶ to about 10¹² EID₅₀ per milliliter ofthe resulting vaccine composition.
 12. A method of claim 10 or 11wherein the mixing step (c) occurs immediately following the contactingstep (b).
 13. A method according to any one of claims 1 to 9 wherein thecontacted composition is transported or stored at ambient temperature.14. A composition comprising a deactivated swine flu virus having aninfectious component and a plurality of surface antigens in contact witha formaldehyde donor agent having a molecular weight that is less thanabout 400 g/mol.
 15. A composition of claim 14 wherein the virusmanufactured by growing the virus in a chicken egg.
 16. A composition ofclaim 14 or 15 wherein the formaldehyde donor agent is selected from anon-crosslinking chemical fixative that contains urea.
 17. A compositionof any one of claims 14 to 16 wherein the formaldehyde donor agent isselected from diazolidinyl urea (DU), imidazolidinyl urea (IDU), or amixture thereof.
 18. A composition of any one of claims 14 to 17 whereinthe formaldehyde donor agent is present in a concentration of less thanabout 1 w/v (grams per 100 ml total volume) or less.
 19. A compositionof any one of claims 14 to 18 wherein the composition can be transportedand stored at ambient temperatures.
 20. A method comprising the stepsof: a) providing a live swine flu virus having an infectious componentand a plurality of surface antigens; b) contacting the swine flu viruswith a formaldehyde donor agent having a molecular weight that isgreater than about 50 g/mol and less than about 400 g/mol for a periodof time sufficient for de-activating the infectious component with theformaldehyde donor agent, and for preserving at least a portion of thesurface antigens to form a deactivated virus c) transporting or storingthe contacted composition at ambient temperature.